Cadherin-11 EC1 Domain Antagonists for Treating Inflammatory Joint Disorders

ABSTRACT

The present invention relates to Cadherin-11 antagonists and compositions comprising Cadherin-11 antagonists. The invention also relates to methods for treating inflammatory joint disorders, such as rheumaotid arthritis, in a mammalian subject by administering a therapeutically effective amount of a Cadherin-11 antagonist.

RELATED APPLICATION(S)

This application claims the benefit of U.S. Provisional Application No.61/010,734, filed on Jan. 11, 2008. The entire teachings of the aboveapplication are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Patients with advanced chronic joint inflammation suffer from severejoint deterioration including bone and cartilage destruction, resultingin long-term pain, deformity, loss of joint function, reduced mobilityand shortened life expectancy.

Joint inflammation is associated with an increased number of cells andinflammatory substances in the joint, which cause irritation, wearingdown of cartilage and swelling of the joint lining. Several differentautoimmune disorders are known to trigger inappropriate or misdirectedinflammation in a joint, resulting in chronic inflammation in the jointsof individuals who suffer from these disorders. Common inflammatoryjoint disorders include rheumatoid arthritis, psoriatic arthritis,Reiter's syndrome and ankylosing spondylitis.

Rheumatoid arthritis (RA) is the most common form of inflammatoryarthritis and is estimated to affect approximately 1 percent of the U.S.population, or about 2.1 million Americans. RA is a chronic disease thatis characterized by inflammation of the lining, or synovium, of thejoints, and can lead to significant bone and cartilage damage over time.RA is more common in women than in men and as many as 3% of women maydevelop rheumatoid arthritis in their lifetime. Currently, the cause ofRA is unknown.

RA can lead to long-term joint damage, resulting in chronic pain, lossof function and disability. In addition, recent research indicates thatpeople with RA, particularly those whose disease is not well controlled,may have a higher risk for heart disease and stroke. Thus, RA is a majornational health burden and there is an urgent need to develop new agentsfor the prevention and treatment of rheumatoid arthritis, and otherinflammatory joint disorders.

SUMMARY OF THE INVENTION

The present invention encompasses, in one embodiment, a Cadherin-11antagonist that specifically binds an extracellular 1 (EC1) domain of amammalian Cadherin-11 protein, and inhibits aggregation of cells thatexpress the mammalian Cadherin-11. In a particular embodiment, theCadherin-11 antagonist is an antibody or an antibody fragment. Inanother embodiment, the Cadherin-11 antagonist is a fusion protein thatcomprises the EC1 domain of a Cadherin-11 protein (e.g., SEQ ID NO:3).

In an additional embodiment, the invention relates to methods oftreating an inflammatory joint disorder in a mammalian subject (e.g., ahuman). The method comprises administering to the mammalian subject atherapeutically effective amount of a Cadherin-11 antagonist of theinvention, thereby resulting in a desired therapeutic effect in themammal. In a particular embodiment, the methods of the invention can beused to treat rheumatoid arthritis.

In another embodiment, the invention encompasses a pharmaceuticalcomposition comprising a Cadherin-11 antagonist of the invention and apharmaceutically-acceptable carrier. In one embodiment, thepharmaceutical composition further comprises a second agent, such as adisease-modifying anti-rheumatic drug or an anti-inflammatory agent.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a Western blot showing detection of a Cadherin-11-EC1-5-Fcfusion protein using anti-Cad-11 antibodies 23C6, 13C2 and 27F3 (seesolid arrows). These antibodies did not recognize the Cadherin-1′-EC1-Fcand Cadherin-11-EC1/2-Fc fusion proteins that were also present on themembrane (see open arrows for positions of the Cadherin-11-EC1-Fc andCadherin-11-EC1/2-Fc proteins on the blot).

FIG. 1B is a graph depicting the binding of public Cadherin-11antibodies 13C2, 23C6 and 5F82 to human Cad-11-EC1-5-Fc fusion protein,but not the Cad-11-EC1-Fc fusion protein, as determined by ELISA. Incontrast, the EC1 antibody H1M1 binds both Cad-11-EC1-Fc fusion proteinand the Cad-11-EC1-5-Fc fusion protein.

FIG. 2 is an amino acid sequence alignment of the first 34 amino acidsof the EC1 domains of human Cad-11 (SEQ ID NO:3), MN-Cad (SEQ ID NO:4),and Cad-8 (SEQ ID NO:5) that are involved in cadherin binding. Donorsequences containing residues that extend into the pocket of a cadherincounter-receptor are indicated by underlining of the left half ofsequence and residues of the pocket sequence are indicated by theunderlining of the right half of sequence in SEQ ID NO:3.

FIG. 3 is a graph depicting the binding of a Cadherin-11-binding Fab toa human Cad-11 EC1 domain peptide as well as the Cad-11-EC1-Fc fusionprotein, but not the Cad-8 or MN-Cad EC1 domain peptides, as determinedby ELISA. Clone 7 demonstrated significant binding to the Cad-11 EC1domain peptide and fusion protein, but not the MN-Cad or Cad-8 EC1domain peptides.

FIG. 4 is a graph depicting data from an in vitro Cad-11 cellaggregation assay. Cad-11 antagonists added to the media, such as a Fabmade from the anti-Cad-11 antibody 13C2, or varying concentrations of ananti-Cadherin-11 EC1 Fab directed to the first 35 amino acids of the EC1domain of Cadherin-11 (designated EC1 Fab clone 7), block Cad-11mediated 431-D-11 cell aggregation. The anti-Cadherin-11 EC1 Fab (clone7) inhibited aggregation of A-431-D-11 epidermoid carcinoma cells at allconcentrations tested in a range of 0.3 μg/ml to 10 μg/ml. In contrast,the Fab made from the 13C2 anti-Cadherin-11 antibody only inhibited cellaggregation at a concentration of 10 μg/ml.

FIG. 5 is a graph depicting data from a second in vitro Cad-11 cellaggregation assay. Percent aggregation of 431-D-11 cells is shown at 40min. after addition of either SME media (designated control), varyingconcentrations of a fusion protein comprising the EC1 domain of Cad-11fused to the human IgG2 hinge, CH2 and CH3 domains (designatedCad-11-EC1-Fc), varying concentrations of an anti-Cadherin-11 EC1 Fabdirected to the first 35 amino acids of the EC1 domain of Cadherin-11(designated Cad-11 EC1 Fab) or varying concentrations of a controlanti-green fluorescent protein (anti-GFP) Fab (designated GFP fAb). Theanti-Cadherin-11 EC1 Fab (clone 7) inhibited aggregation of Cad-11expressing 431-D-11 cells at concentrations of 3 μg/ml, 1 μg/ml and 0.1μg/ml. The EC1-Fc fusion protein inhibited aggregation of 431-D-11 cellsat concentrations of 3 μg/ml. In contrast, the anti-GFP Fab failed toinhibit cell aggregation significantly at any of the testconcentrations.

FIG. 6 is a graph depicting inhibition of Cad-11 mediated cellaggregation by various anti-Cadherin-11 EC1 Fabs that have bindingspecificity for Cad-11 alone (EC1 fAb clone 7 and clone 4), Cad-11 andCad-8 (EC1 fAb clone 6), or Cad-11 and MN-Cad (EC1 fAb clone 5), usingan in vitro cell aggregation assay. All Fabs tested inhibited 431-D-11cell aggregation relative to the control (D-11 SME; left bar).

FIG. 7 is a graph depicting inhibition of Cad-11 mediated cellaggregation by anti-Cad-11 Fabs that have binding specificity for Cad-11alone (EC1 fAb clone 7), or Cad-11 and MN-Cad (EC1 fAb clone 8), usingan in vitro cell aggregation assay. The specificity of the Fabs testedis shown in parentheses next to each Fab designation. Bothcadherin-specific Fabs inhibited cell aggregation (middle and rightbars) relative to a control Fab that was specific for GFP (left bar).

FIG. 8 shows the nucleotide (DNA) sequence (SEQ ID NO:6) of the humanCad-11-EC1-hIgG2-Fc1 fusion protein (Cad-11-EC1-Fc). The sequence of thehuman Cadherin-11 extracellular domain is shown in italics, the BglIIsite is underlined, and the sequence encoding the human IgG₂-Fc1 regionis shown in bold lettering.

FIG. 9 shows the amino acid sequence (SEQ ID NO:7) of the humanCad-11-EC1-hIgG2-Fc1 fusion protein (Cad-11-EC1-Fc). The sequence of thehuman Cadherin-11 extracellular domain is shown in italics, the sequenceencoded by the BglII site is underlined, and the sequence of the humanIgG₂-Fc1 region is shown in bold lettering.

FIG. 10 is an image of an SDS polyacrylamide gel that has been stainedwith Coomassie Blue, which shows the predominant intense bandscorresponding to the monomeric form of the purified Cad-11-EC1-hIgG₂-Fc1(middle lane) and Cad-11-EC1/2-hIgG₂-Fc1 (right lane) fusion proteins,respectively, following purification from cell culture medium using aprotein A column. Molecular weight standards are shown in the left lane.

FIG. 11 is a Western blot showing the detection of humanCad-11-EC1-hIgG₂-Fc1 (middle lane) and Cad-11-EC1/2-hIgG₂-Fc1 (rightlane) fusion proteins using an anti-human IgG antibody conjugated tohorse radish peroxidase (HRP). The predominant observed band in eachlane corresponds to the locations of the monomeric forms of the fusionproteins. The locations of the dimeric forms of the fusion proteins arealso visible (see less intense higher molecular weight bands), due toincomplete reducing conditions. Molecular weight standards are shown inthe left lane.

FIG. 12 is a graph depicting that a Cad-11-EC1-Fc fusion protein and amouse anti-Cad-11 antibody, 13C2, inhibit the invasion of Cad-11expressing human fibroblast-like synoviocytes into a matrigel plug atthe indicated concentrations compared to untreated cells, labeledInvasion. Data is pooled from two independent experiments.

FIGS. 13A and B are graphs depicting data from two in vitro Cad-11 cellaggregation assays. Percent aggregation of Cad-11 expressing 431-D-11cells is shown at 40 min. after addition of either SME media (designatedcontrol) or a Cad-11 fusion protein. FIG. 13A shows the inhibition ofaggregation in the presence of varying concentrations of a fusionprotein comprising the 5 extracellular domains of Cad-11 fused to thehuman IgG2 hinge, CH2 and CH3 domains (designated Cad-11-EC1-5-Fc). FIG.13B shows the inhibition of aggregation of varying concentrations of afusion protein comprising either the N-terminal extracellular domain(EC1 domain) of Cad-11 fused to the human IgG2 hinge, CH2 and CH3domains (designated Cad-11-EC1-Fc) or Cad-11-EC1-5-Fc.

FIGS. 14A-C show the human Cadherin-11 cDNA sequence (SEQ ID NO:1; seeGenbank Accession No. NM001797).

FIG. 15 shows the human Cadherin-11 protein sequence (SEQ ID NO:2; seeGenbank Accession No. NP001788).

FIG. 16 is a graph depicting the level of binding of antibodies in mediafrom peptide 4 hybridomas (HL), or control hybridoma media (Media), toproteins containing the EC1-2 domains of Cad-11, Cad-8 or MN-Cad, asdetermined by ELISA.

FIGS. 17A-C are representative graphs depicting the intensity of cellstaining (MFI; mean fluorescence intensity) as a measure of binding ofH14 antibody to Cad-11-expressing 431-D-11 cells.

FIGS. 17D-F are representative graphs depicting the absence of 431-Dcell staining (MFI; mean fluorescence intensity) relative to FIGS.17A-C, indicating a lack of binding of H14 antibody to the Cad-11negative cells.

FIGS. 17G-I are representative graphs depicting the intensity of cellstaining (MFI; mean fluorescence intensity) as a measure of binding ofH1M1 antibody to Cad-11-expressing 431-D-11 cells.

FIG. 18A is a graph depicting the binding of H14 antibody toCad-11-expressing cells, and the absence of H14 binding to Cad-11negative control cells, at varying concentrations of antibody, asmeasured by the intensity of cell staining (MFI; mean fluorescenceintensity).

FIG. 18B is a graph depicting the binding of H1M1 antibody toCad-11-expressing cells, and the absence of binding of H1M1 to Cad-11negative control cells, at varying concentrations of antibody, asmeasured by the intensity of cell staining (MFI; mean fluorescenceintensity).

FIG. 19A is a graph depicting the degree of binding of the H14anti-Cad-11 antibody to Cad-11 and Cad-8 EC1 domain peptides at variousantibody concentrations, as determined by ELISA.

FIG. 19B is a graph depicting the absence of binding of the H14anti-Cad-11 antibody to Cad7, MNCad, Cad9, Cad18, Cad20 or Cad24 EC1domain peptides at various antibody concentrations, as determined byELISA.

FIG. 20 is a graph depicting the binding of the H1M1 anti-Cad-11antibody to Cad-11 and Cad-8 EC1 domain peptides at varying antibodyconcentrations, as determined by ELISA.

FIG. 21A is a graph depicting the degree of binding of the H1M1anti-Cad-11 antibody to various Cad-11 EC1 domain peptide immunogens(PEP1, PEP2, PEP3 and PEP4), as well as the Cad-11 EC1 domain fusionprotein (EFL) and human IgG control (Fc block), as determined by ELISA.

FIG. 21B is a graph depicting the degree of binding of the H14anti-Cad-11 antibody to various Cad-11 EC1 domain peptide immunogens(PEP1, PEP2, PEP3 and PEP4), as well as the Cad-11 EC1 domain fusionprotein (EFL) and human IgG control (Fc block), as determined by ELISA.

FIG. 22 is a schematic diagram depicting the sequence of the first 37amino acids of the EC1 domain of human Cadherin-11 and the portions ofthis sequence encompassed by each of Peptides 1-4. Amino acid residuesshared by Peptides 2 and 4 that are upstream of Peptide 3 arehighlighted in the boxed region. Amino acids directly involved in Cad-11to Cad-11 binding are underlined.

FIG. 23A is a photograph showing a large mass of aggregatedCad-11-expressing cells that were treated with a control isotypeantibody.

FIG. 23B is a photograph showing small clumps of H1M1-treatedCad-11-expressing cells that did not progress to form the large massesobserved in FIG. 23A.

FIG. 23C is a photograph showing that untreated parental Cad-11 negativecells remain as groups of single or double cells.

FIG. 24A is a photograph depicting a culture of Cad-11 expressing cellswith large masses of aggregated cells.

FIG. 24B is a photograph depicting a culture of Cad-11 expressing cellswith predominantly single cells with small and infrequent cell clustersrelative to those shown in FIG. 24A following treatment with the H14Cad-11 EC1 domain antibody.

FIG. 25 is a graph depicting inhibition of arthritis-associated jointswelling in mice treated with increasing dosages of H1M1 anti-Cad-11antibody relative to untreated control mice.

FIG. 26 is a graph depicting inhibition of arthritis-associated jointswelling in mice treated with 0.3 mg of either H14 or H1M1 anti-Cad-11antibodies every second day relative to untreated control mice.

FIG. 27 is a graph showing that treatment with 0.3 mg of either H1M1 orH14 antibody delayed the development of arthritis in a mouse modelcompared to an untreated control.

FIG. 28 is a graph depicting the degree of binding ofantibody-containing media from peptide 3 hybridomas (HL), or controlhybridoma media (Media), to the EC1-2 domains of Cad-11, Cad-8, andMN-Cadherin, or a Cad-11 EC1-Fc fusion protein.

FIG. 29 is a graph depicting the degree of binding of anti-Cad-11antibodies from the peptide 3 hybridomas to cells expressing humanCad-11 protein (see arrow) and non-Cad-11-expressing control cells thatexpressed Neos.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the terms “Cadherin-11,” “Cad-11,” and “OB-Cadherin”refer to a naturally occurring or endogenous Cadherin-11 (e.g.,mammalian, for example human) protein, and to proteins having an aminoacid sequence that is the same as that of naturally occurring orendogenous Cadherin-11 protein (e.g., recombinant proteins, syntheticproteins). Accordingly, the terms “Cadherin-11,” “Cad-11,” and“OB-Cadherin,” which are used interchangeably herein, includepolymorphic or allelic variants and other isoforms of a Cadherin-11protein (e.g., mammalian, human) produced by, e.g., alternative splicingor other cellular processes, that occur naturally in mammals (e.g.,humans, non-human primates). Preferably, the Cadherin-11 protein is ahuman protein that has the amino acid sequence of SEQ ID NO:2 (See,Genbank Accession No. NP001788 and FIG. 15).

As defined herein, a “Cadherin-11 antagonist” is an agent (e.g.,antibody, fusion protein, peptide, peptidomimetic, small molecule,nucleic acid) that specifically binds an EC1 domain of a Cadherin-11protein and inhibits (e.g., reduces, prevents) one or moreCadherin-11-mediated activities in a cell. Cadherin-11-mediatedactivities include, but are not limited to, binding of a Cadherin-11protein to one or more other Cadherin-11 proteins in a homotypicfashion, aggregation of cells that express Cadherin-11, induction ofenzyme (e.g., collagenase, serine proteases, MMP1, MMP3, MMP13)expression or activity, and induction of cytokines or growth factors(e.g., IL-6, IL-8 or RANKL or TRANCE). In one embodiment, theCadherin-11 antagonist can inhibit the binding of a Cadherin-11 proteinto one or more other Cadherin-11 proteins by, for example, blocking theinteraction between the donor sequences in the EC1 domain of a Cad-11protein (e.g., a Cad-11 protein expressed on the surface of a cell) withthe pocket sequence in the EC1 domain of one or more other Cad-11proteins (e.g., one or more Cad-11 proteins expressed on the surface ofanother cell).

As used herein, a Cadherin-11 antagonist that “specifically binds” anEC1 domain of a Cadherin-11 protein refers to a Cadherin-11 antagonistthat binds (e.g., under physiological conditions) an EC1 domain of aCadherin-11 protein with an affinity (e.g., a binding affinity) that isat least about 5 fold, preferably at least about 10 fold, greater thanthe affinity with which the Cadherin-11 antagonist binds an EC1 domainof another cadherin protein (e.g., MN-Cadherin, Cadherin-8). In aparticular embodiment, the Cadherin-11 antagonist that specificallybinds an EC1 domain of a Cadherin-11 protein binds an epitope present inSEQ. ID NO:3, the N-terminal portion of the EC1 domain of humanCadherin-11, with an affinity that is at least about 5 fold, preferablyat least about 10 fold, greater than the affinity with which theCadherin-11 antagonist binds an epitope present in SEQ ID NO:4, theN-terminal portion of the EC1 domain of human MN-Cadherin, and theaffinity with which the Cadherin-11 antagonist binds an epitope presentin SEQ ID NO:5, the N-terminal portion of the EC1 domain of humanCadherin-8.

As used herein, the term “antibody” is intended to encompass both wholeantibodies and antibody fragments (e.g., antigen-binding fragments ofantibodies, for example, Fv, Fc, Fd, Fab, Fab′, F(ab′), and dAbfragments). “Antibody” refers to both polyclonal and monoclonalantibodies and includes naturally-occurring and engineered antibodies.Thus, the term “antibody” includes, for example, human, chimeric,humanized, primatized, veneered, single chain, and domain antibodies(dAbs). (See e.g., Harlow et al., Antibodies A Laboratory Manual, ColdSpring Harbor Laboratory, 1988).

The term “epitope” refers to a unit of structure conventionally bound byan immunoglobulin V_(H)/V_(L) pair. An epitope defines the minimumbinding site for an antibody, and thus represent the target ofspecificity of an antibody.

The term “fusion protein” refers to a naturally occurring, synthetic,semi-synthetic or recombinant single protein molecule that comprises allor a portion of two or more heterologous polypeptides

The term “polypeptide” refers to a polymer of amino acids, and not to aspecific length; thus, peptides, oligopeptides and proteins are includedwithin the definition of a polypeptide.

As used herein, the term “peptide” refers to a compound consisting offrom about 2 to about 100 amino acid residues wherein the amino group ofone amino acid is linked to the carboxyl group of another amino acid bya peptide bond. Such peptides are typically less than about 100 aminoacid residues in length and preferably are about 10, about 20, about 30,about 40 or about 50 residues.

As used herein, the term “peptidomimetic” refers to molecules which arenot peptides or proteins, but which mimic aspects of their structures.Peptidomimetic antagonists can be prepared by conventional chemicalmethods (see e.g., Damewood J. R. “Peptide Mimetic Design with the Aidof Computational Chemistry” in Reviews in Computational Biology, 2007,Vol. 9, pp. 1-80, John Wiley and Sons, Inc., New York, 1996; KazmierskiW. K., “Methods of Molecular Medicine: Peptidomimetic Protocols,” HumanaPress, New Jersey, 1999).

As defined herein, “therapy” is the administration of a particulartherapeutic or prophalytic agent to a subject (e.g., a mammal, a human),which results in a desired therapeutic or prophylactic benefit to thesubject.

As defined herein a “treatment regimen” is a regimen in which one ormore therapeutic or prophalytic agents are administered to a mammaliansubject at a particular dose (e.g., level, amount, quantity) and on aparticular schedule or at particular intervals (e.g., minutes, days,weeks, months).

As defined herein, a “therapeutically effective amount” is an amountsufficient to achieve the desired therapeutic or prophylactic effectunder the conditions of administration, such as an amount sufficient toinhibit (i.e., reduce, prevent) inflammation in a joint (e.g., byinhibiting the aggregation of cells, for example synoviocytes, thatexpress Cadherin-11). The effectiveness of a therapy (e.g., thereduction of inflammation in a joint and/or prevention of inflammationin a joint) can be

determined by suitable methods (e.g., imaging methods, such as MRI, NMR,CT).

Cadherins

Cadherins belong to a large family of Ca²⁺-dependent adhesion moleculesthat mediate cell adhesion by binding to other cadherins in a homotypicmanner (M J Wheelock and K R Johnson, Ann. Rev. Cell Dev. Biol. 19:207-235 (2003). Classical cadherins are single-pass transmembraneproteins that contain five extracellular cadherin (EC) domains, eachapproximately 110 amino acids in length, a transmembrane region and aconserved cytoplasmic domain. Cadherins are divided into either type Ior type II cadherins based on the degree of homology between the ECdomains. Type II cadherins include human cadherins-5, -6, -8, -11, and-12, and MN-cadherin. The relative importance of the role of each of theextracellular domains in mediating inter-cellular binding is unclear.

Cadherin-11 Activity in Synoviocytes

Cadherin-11 mediates synoviocyte to synoviocyte binding in the synoviallining of articulated joints (Valencia et al., J. Exp. Med.200(12):1673-1679 (2004); Kiener and Brenner, Arthritis Res Ther.7(2):49-54 (2005)). A fusion protein that comprised all fiveextracellular cadherin domains of human Cadherin-11, fused to thehinge-CH2-CH3 domain of human IgG₂, inhibited synoviocyte liningformation in vitro (Kiener et al., Am. J. Pathol. 168 (2006)). Inaddition, antagonistic anti-Cadherin-11 antibodies and a fusion proteinthat comprised EC1-5 of murine Cadherin-11, fused to the hinge-CH₂—CH₃domains of murine IgG2a, inhibited inflammation and joint swelling inmurine models of rheumatoid arthritis (Lee et al., Science 315:1006-1010(2007)).

Cadherin-11 Antagonists

A Cadherin-11 antagonist of the invention can be any agent thatspecifically binds an EC1 domain of a Cadherin-11 protein and inhibits(e.g., reduces, prevents) one or more Cadherin-11-mediated activities ina cell. Cadherin-11-mediated activities include, but are not limited to,aggregation of cells that express Cadherin-11 on the cell surface, andexpression or secretion of factors such as, for example, collagenase,serine proteases, MMP1, MMP3, IL-6, IL-8 or RANKL/TRANCE. The agent canbe an antibody, a fusion protein, a peptide, a peptidomimetic, a smallmolecule, or a nucleic acid, among others.

Cadherin-11 Antibodies

As described herein, antibodies that bind an epitope within anN-terminal portion of the EC1 domain of human Cadherin-11 that comprisesthe donor sequences and cadherin-binding pocket of Cad-11 (e.g., SEQ IDNO:3), block Cadherin-11 activity in vitro more effectively thanantibodies that bind to epitopes in other regions of this protein (SeeExamples 1 and 2).

Accordingly, in one embodiment, the invention provides an antibody orantigen-binding fragment thereof that binds (e.g., specifically binds)an epitope that is present in the N-terminal portion of the EC1 domainof a Cadherin-11 protein that comprises the donor sequences andcadherin-binding pocket of Cad-11. The term “antibody” is intended toencompass all types of polyclonal and monoclonal antibodies (e.g.,human, chimeric, humanized, primatized, veneered, single chain, domainantibodies (dAbs)) and antigen-binding fragments of antibodies (e.g.,Fv, Fc, Fd, Fab, Fab′, F(ab′), dAb). (See e.g., Harlow et al.,Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Ina particular embodiment, the Cad-11 EC1 domain-specific antibody is ahuman antibody or humanized antibody. Cad-11 EC1 domain-specificantibodies can also be directly or indirectly linked to a cytotoxicagent.

Other antibodies or antibody fragments that specifically bind to anN-terminal portion of the EC1 domain of a Cad-11 protein and inhibit theactivity of the Cad-11 protein can also be produced, constructed,engineered and/or isolated by conventional methods or other suitabletechniques. For example, antibodies which are specific for the EC1domain of a Cadherin-11 protein can be raised against an appropriateimmunogen, such as a recombinant mammalian (e.g., human) Cadherin-11 EC1domain peptide (e.g., SEQ ID NO:3) or a portion thereof (includingsynthetic molecules, e.g., synthetic peptides). A variety of methodshave been described (see e.g., Kohler et al., Nature, 256: 495-497(1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature266: 550-552 (1977); Koprowski et al., U.S. Pat. No. 4,172,124; Harlow,E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory: Cold Spring Harbor, N.Y.); Current Protocols InMolecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F. M. etal., Eds., (John Wiley & Sons: New York, N.Y.), Chapter 11, (1991)).Antibodies can also be raised by immunizing a suitable host (e.g.,mouse) with cells that express the EC1 domain of Cadherin-11 (e.g.,cancer cells/cell lines) or cells engineered to express the EC1 domainof Cadherin-11 (e.g., transfected cells). (See e.g., Chuntharapai etal., J. Immunol., 152:1783-1789 (1994); Chuntharapai et al. U.S. Pat.No. 5,440,021). For the production of monoclonal antibodies, a hybridomacan be produced by fusing a suitable immortal cell line (e.g., a myelomacell line such as SP2/0 or P3X63 Ag8.653) with antibody producing cells.The antibody producing cells can be obtained from the peripheral blood,or preferably, the spleen or lymph nodes, of humans or other suitableanimals immunized with the antigen of interest. The fused cells(hybridomas) can be isolated using selective culture conditions, andcloned by limited dilution. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

Antibody fragments can be produced by enzymatic cleavage or byrecombinant techniques. For example, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Other proteases withthe requisite substrate specificity can also be used to generate Fab orF(ab′)₂ fragments. Antibodies can also be produced in a variety oftruncated forms using antibody genes in which one or more stop codonshas been introduced upstream of the natural stop site. For example, achimeric gene encoding a F(ab′)₂ heavy chain portion can be designed toinclude DNA sequences encoding the CH₁ domain and hinge region of theheavy chain. Single chain antibodies, and human, chimeric, humanized orprimatized (CDR-grafted), or veneered antibodies, as well as chimeric,CDR-grafted or veneered single chain antibodies, comprising portionsderived from different species, and the like are also encompassed by thepresent invention and the term “antibody”. The various portions of theseantibodies can be joined together chemically by conventional techniques,or can be prepared as a contiguous protein using genetic engineeringtechniques. For example, nucleic acids encoding a chimeric or humanizedchain can be expressed to produce a contiguous protein. See, e.g.,Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European PatentNo. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO 86/01533;Neuberger, M. S. et al., European Patent No. 0,194,276 B1; Winter, U.S.Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 B1; Queen etal., European Patent No. 0 451 216 B1; and Padlan, E. A. et al., EP 0519 596 A1. See also, Newman, R. et al., BioTechnology, 10: 1455-1460(1992), regarding primatized antibody, and Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988))regarding single chain antibodies.

Humanized antibodies can be produced using synthetic or recombinant DNAtechnology using standard methods or other suitable techniques. Nucleicacid (e.g., cDNA) sequences coding for humanized variable regions canalso be constructed using PCR mutagenesis methods to alter DNA sequencesencoding a human or humanized chain, such as a DNA template from apreviously humanized variable region (see e.g., Kamman, M., et al.,Nucl. Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research,53: 851-856 (1993); Daugherty, B. L. et al., Nucleic Acids Res., 19(9):2471-2476 (1991); and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302(1991)). Using these or other suitable methods, variants can also bereadily produced. In one embodiment, cloned variable regions (e.g.,dAbs) can be mutated, and sequences encoding variants with the desiredspecificity can be selected (e.g., from a phage library; see e.g.,Krebber et al., U.S. Pat. No. 5,514,548; Hoogenboom et al., WO 93/06213,published Apr. 1, 1993).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, for example, methods whichselect a recombinant antibody or antibody-binding fragment (e.g., dAbs)from a library (e.g., a phage display library), or which rely uponimmunization of transgenic animals (e.g., mice). Transgenic animalscapable of producing a repertoire of human antibodies are well-known inthe art (e.g., Xenomouse® (Abgenix, Fremont, Calif.)) and can beproduced using suitable methods (see e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90: 2551-2555 (1993); Jakobovits et al., Nature,362: 255-258 (1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani etal., U.S. Pat. No. 5,545,807; Lonberg et al., WO 97/13852).

The invention encompasses, in one embodiment, a Cad-11 antibody thatbinds to an epitope that is present in the first about 37 amino acids ofthe EC1 domain of human Cad-11 (SEQ ID NO: 13). In a particularembodiment, the invention relates to a Cad-11 antibody that binds to anepitope that is present in SEQ ID NO:10. In a further embodiment, theinvention relates to a Cad-11 antibody that binds to an epitope thatcomprises SEQ ID NO:11. In another embodiment, the invention relates toa Cad-11 antibody that binds to an epitope that is present in SEQ IDNO:12.

In one embodiment, the invention relates to a Cad-11 antibody producedby hybridoma H1M1 (ATCC accession number ______), having been depositedon Jan. 8, 2009 at the American Type Culture Collection (ATCC), P.O. Box1549, Manassas, Va. 20108, United States of America. In anotherembodiment, the invention provides a Cad-11 antibody produced byhybridoma H14 (ATCC accession number ______), having been deposited onJan. 8, 2009 at the American Type Culture Collection (ATCC), P.O. Box1549, Manassas, Va. 20108, United States of America.

The invention also encompasses antibodies that specifically compete witha Cad-11 antibody produced by hybridoma H1M1 and/or a Cad-11 antibodyproduced by hybridoma H14 for binding to a human Cad-11 protein or anEC1-domain containing portion thereof (e.g., SEQ ID NO:3, 10, 12, 13).In a particular embodiment, an antibody that specifically competes witha Cad-11 antibody produced by hybridoma H1M1 and/or hybridoma H14 blocks(e.g., inhibits, diminishes, prevents) the binding of a Cad-11 antibodyproduced by hybridoma H1M1 and/or hybridoma H14 to a human Cad-11protein or EC1-domain containing portion thereof (e.g., SEQ ID NO:3, 10,12, 13).

In addition, the invention encompasses antibodies having a bindingaffinity for a human Cad-11 protein or EC1-domain containing portionthereof (e.g., SEQ ID NO:3, 10, 12, 13) that is at least as great as thebinding affinity of a Cad-11 antibody produced by hybridoma H1M1 and/ora Cad-11 antibody produced by hybridoma H14 for a human Cad-11 proteinor EC1-domain containing portion thereof.

Cadherin-11 Fusion Proteins

In addition, immunoglobulin fusion proteins that contain only the EC1domain of human Cad-11 (e.g., the EC1 domain of human Cad-11 fused to aportion of human IgG) inhibited Cad-11 activity in vitro moreeffectively than a fusion protein that included a larger portion of theEC region of Cad-11, which contained all 5 EC domains.

Cadherin-11 antagonists also encompass chimeric, or fusion, proteinsthat comprise at least about the N-terminal 35 amino acids of the EC1domain of human Cad-11 (SEQ ID NO:2) operatively linked to all or aportion of a heterologous protein. “Operatively linked” indicates thatthe portion of the Cad-11 EC1 domain and the heterologous protein arefused in-frame. The heterologous protein can be fused to the N-terminusor C-terminus of the protein. For example, the fusion protein can be aGST-fusion protein in which the protein sequences are fused to theC-terminus of a GST sequence. Other types of fusion proteins include,but are not limited to, enzymatic fusion proteins, for example,β-galactosidase fusion proteins, yeast two-hybrid GAL fusion proteins,poly-His fusions, FLAG-tagged fusion proteins, GFP fusion proteins, andimmunoglobulin (Ig) fusion proteins. Such fusion protein can facilitatepurification (e.g., of a recombinant fusion protein). In certain hostcells (e.g., mammalian host cells), expression and/or secretion of aprotein can be increased by using a heterologous signal sequence.Therefore, in another embodiment, the fusion protein contains aheterologous signal sequence at its N-terminus.

EP-A-0 464 533 discloses fusion proteins comprising various portions ofimmunoglobulin constant regions. The Fc is useful in therapy anddiagnosis and thus results, for example, in improved pharmacokineticproperties (see, for example, EP-A 0232 262). In drug discovery, forexample, human proteins have been fused with Fc portions for the purposeof high-throughput screening assays to identify antagonists (Bennett etal., Journal of Molecular Recognition 8:52-58 (1995); Johanson et al.,J. Biol. Chem., 270(16):9459-9471 (1995)). Thus, this invention alsoencompasses soluble fusion proteins containing a protein Cad-11antagonist of the invention and various portions of the constant regionsof heavy and/or light chains of immunoglobulins of various subclasses(e.g., IgG, IgM, IgA, IgE). Advantages of immunoglobulin fusion proteinsof the present invention include one or more of the following: (1)increased avidity for multivalent ligands due to the resulting bivalencyof dimeric fusion proteins, (2) longer serum half-life, (3) the abilityto activate effector cells via the Fc domain, (4) ease of purification(for example, by protein A chromatography), (5) affinity for Cad-11 and(6) the ability to block Cad-11 mediated activity.

Accordingly, in particular embodiments, the Cad-11 antagonist is afusion protein that comprises a portion of the extracellular region of aCadherin-11 protein that includes an N-terminal portion of the EC1domain (amino acids 54-90 of SEQ ID NO:2), operatively linked to all, ora portion of, a mammalian immunoglobulin protein. In a particularembodiment, the immunoglobulin fusion proteins of the invention do notcomprise a portion of the extracellular region of Cadherin-11 thatincludes all five EC domains that are contained within amino acids 1-609of SEQ ID NO:2. In certain embodiments, the portion of the humanCadherin-11 extracellular region can include, for example, amino acids1-160, amino acids 1-259 or amino acids 1-269 of SEQ ID NO:2. In aparticular embodiment, the fusion protein lacks the leader andpro-region of human Cadherin-11 (amino acids 1-53 of SEQ ID NO:2) anduses a heterogologous leader sequence. The immunoglobulin portion can befrom any vertebrate source, such as murine, but preferably, is a humanimmunoglobulin protein. In one embodiment, the mammalian immunoglobulinprotein is a human IgG₂ protein or a portion thereof, such as thehinge-CH₂—CH₃ portion of human IgG₂.

A chimeric or fusion protein of the invention can be produced bystandard recombinant DNA techniques. For example, DNA fragments codingfor different protein sequences (e.g., a Cad-11 EC1 domain peptide and amammalian immunoglobulin) are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of nucleic acid fragmentscan be carried out using anchor primers that give rise to complementaryoverhangs between two consecutive nucleic acid fragments that cansubsequently be annealed and re-amplified to generate a chimeric nucleicacid sequence (see Ausubel et al., Current Protocols in MolecularBiology, 1992). Moreover, many expression vectors are commerciallyavailable that already encode a fusion moiety (e.g., a GST moiety, an Fcmoiety). A nucleic acid molecule encoding protein Cad-11 antagonist canbe cloned into such an expression vector that the fusion moiety (e.g.,immunoglobulin) is linked in-frame to the protein.

The immunoglobulin fusion proteins of the invention can be provided asmonomers, dimers, tetramers or other multimers (e.g., polymers). Forexample, variable domains of the immunoglobulin portion of the fusionprotein may be linked together to form multivalent ligands by, forexample, provision of a hinge region at the C-terminus of each V domainand disulphide bonding between cysteines in the hinge regions; orprovision of heavy chains each with a cysteine at the C-terminus of thedomain, the cysteines being disulphide bonded together; or production ofV-CH & V-CL to produce a Fab format; or use of peptide linkers (forexample Gly₄Ser linkers) to produce dimers, trimers and furthermultimers. For example, such ligands can be linked to an antibody Fcregion comprising one or both of C_(H)2 and C_(H)3 domains, andoptionally a hinge region. For example, vectors encoding ligands linkedas a single nucleotide sequence to an Fc region may be used to preparesuch ligands (e.g., by expression).

The immunoglobulin fusion proteins of the invention can be conjugated toother moieties including, but not limited to, multimers of polyetheleneglycol (PEG) or its derivatives (e.g., poly methyl ethylene glycol),radionuclides, cytotoxic agents and drugs, and subsequently used for invivo therapy. Examples of radionuclides include ²¹²Bi, ¹³¹I, ¹⁸⁶Re, and⁹⁰Y, among others. The radionuclides exert their cytotoxic effect bylocally irradiating the cells, leading to various intracellular lesions,as is known in the art of radiotherapy. Cytotoxic drugs that can beconjugated to the fusion proteins include, but are not limited to,daunorubicin, doxorubicin, methotrexate, and Mitomycin C. Cytotoxicdrugs interfere with critical cellular processes including DNA, RNA, andprotein synthesis. For a fuller exposition of these classes of drugs,which are known in the art, and their mechanisms of action, see Goodman,A. G., et al., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 8th Ed., Macmillan Publishing Col, 1990. Katzung, ed.,Basic and Clinical Pharmacology, Fifth Edition, p 768-769, 808-809, 896,Appleton and Lange, Norwalk, Conn.

As used herein, the term “immunoglobulin fusion protein” includesfragments of the immunoglobulin fusion proteins of the invention. Suchfragments are intended to be within the scope of this invention. Forexample, once the molecules are isolated, they can be cleaved withprotease to generate fragments that remain capable of binding the EC1domain of human Cad-11.

Peptide Antagonists

The Cadherin-11 antagonist of the invention can also be a peptide thatbinds to the EC1 domain of a Cadherin-11 protein. The peptide cancomprise any suitable L- and/or D-amino acid, for example, commonα-amino acids (e.g., alanine, glycine, valine), non-α-amino acids (e.g.,β-alanine, 4-aminobutyric acid, 6-aminocaproic acid, sarcosine,statine), and unusual amino acids (e.g., citrulline, homocitruline,homoserine, norleucine, norvaline, ornithine). The amino, carboxyland/or other functional groups on a peptide can be free (e.g.,unmodified) or protected with a suitable protecting group. Suitableprotecting groups for amino and carboxyl groups, and methods for addingor removing protecting groups are known in the art and are disclosed in,for example, Green and Wuts, “Protecting Groups in Organic Synthesis”,John Wiley and Sons, 1991. The functional groups of a peptide can alsobe derivatized (e.g., alkylated) using art-known methods.

The peptide Cad-11 antagonist can comprise one or more modifications(e.g., amino acid linkers, acylation, acetylation, amidation,methylation, terminal modifiers (e.g., cyclizing modifications)), ifdesired. The peptide can also contain chemical modifications (e.g.,N-methyl-α-amino group substitution). In addition, the peptideantagonist can be an analog of a known and/or naturally-occurringpeptide, for example, a peptide analog having conservative amino acidresidue substitution(s). These modifications can improve variousproperties of the peptide (e.g., solubility, binding), including itsCadherin-11 antagonist activity.

Cad-11 antagonists that are peptides can be linear, branched or cyclic,e.g., a peptide having a heteroatom ring structure that includes severalamide bonds. In a particular embodiment, the peptide is a cyclicpeptide. Such peptides can be produced by one of skill in the art usingstandard techniques. For example, a peptide can be derived or removedfrom a native protein by enzymatic or chemical cleavage, or can besynthesized by suitable methods, for example, solid phase peptidesynthesis (e.g., Merrifield-type synthesis) (see, e.g., Bodanszky et al.“Peptide Synthesis,” John Wiley & Sons, Second Edition, 1976). Peptidesthat are Cadherin-11 antagonists can also be produced, for example,using recombinant DNA methodologies or other suitable methods (see,e.g., Sambrook J. and Russell D. W., Molecular Cloning: A LaboratoryManual, 3^(rd) Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 2001).

Peptides can be synthesized and assembled into libraries comprising afew to many discrete molecular species. Such libraries can be preparedusing methods of combinatorial chemistry, and can be screened using anysuitable method to determine if the library comprises peptides with adesired biological activity. Such peptide antagonists can then beisolated using suitable methods.

Peptidomimetic Antagonists

Cadherin-11 antagonists can also be peptidomimetics. For example,polysaccharides can be prepared that have the same functional groups aspeptides. Peptidomimetics can be designed, for example, by establishingthe three dimensional structure of a peptide agent in the environment inwhich it is bound or will bind to a target molecule. The peptidomimeticcomprises at least two components, the binding moiety or moieties andthe backbone or supporting structure.

The binding moieties are the chemical atoms or groups which will reactor form a complex (e.g., through hydrophobic or ionic interactions) witha target molecule, for example, with amino acids in the EC1 domain ofCad-11. For example, the binding moieties in a peptidomimetic can be thesame as those in a peptide or protein antagonist. The binding moietiescan be an atom or chemical group which reacts with the receptor in thesame or similar manner as the binding moiety in the peptide antagonist.For example, computational chemistry can be used to design peptidemimetics of the donor sequences of the EC1 domain of a Cadherin-11protein, for instance, which can bind to the pocket sequence in the EC1domain of Cad-11 proteins. Examples of binding moieties suitable for usein designing a peptidomimetic for a basic amino acid in a peptideinclude nitrogen containing groups, such as amines, ammoniums,guanidines and amides or phosphoniums. Examples of binding moietiessuitable for use in designing a peptidomimetic for an acidic amino acidinclude, for example, carboxyl, lower alkyl carboxylic acid ester,sulfonic acid, a lower alkyl sulfonic acid ester or a phosphorous acidor ester thereof.

The supporting structure is the chemical entity that, when bound to thebinding moiety or moieties, provides the three dimensional configurationof the peptidomimetic. The supporting structure can be organic orinorganic. Examples of organic supporting structures includepolysaccharides, polymers or oligomers of organic synthetic polymers(such as, polyvinyl alcohol or polylactide). It is preferred that thesupporting structure possess substantially the same size and dimensionsas the peptide backbone or supporting structure. This can be determinedby calculating or measuring the size of the atoms and bonds of thepeptide and peptidomimetic. In one embodiment, the nitrogen of thepeptide bond can be substituted with oxygen or sulfur, for example,forming a polyester backbone. In another embodiment, the carbonyl can besubstituted with a sulfonyl group or sulfinyl group, thereby forming apolyamide (e.g., a polysulfonamide). Reverse amides of the peptide canbe made (e.g., substituting one or more-CONH-groups for a-NHCO-group).In yet another embodiment, the peptide backbone can be substituted witha polysilane backbone.

These compounds can be manufactured by known methods. For example, apolyester peptidomimetic can be prepared by substituting a hydroxylgroup for the corresponding α-amino group on amino acids, therebypreparing a hydroxyacid and sequentially esterifying the hydroxyacids,optionally blocking the basic and acidic side chains to minimize sidereactions. Determining an appropriate chemical synthesis route cangenerally be readily identified upon determining the chemical structure.

Peptidomimetics can be synthesized and assembled into librariescomprising a few to many discrete molecular species. Such libraries canbe prepared using well-known methods of combinatorial chemistry, and canbe screened to determine if the library comprises one or morepeptidomimetics which have the desired activity. Such peptidomimeticantagonists can then be isolated by suitable methods.

Small Molecule Antagonists

Cadherin-11 antagonists can also be small molecules. Examples of smallmolecules include organic compounds, organometallic compounds, inorganiccompounds, and salts of organic, organometallic or inorganic compounds.Atoms in a small molecule are typically linked together via covalentand/or ionic bonds. The arrangement of atoms in a small organic moleculemay represent a chain (e.g. a carbon-carbon chain or a carbon-heteroatomchain), or may represent a ring containing carbon atoms, e.g. benzene ora policyclic system, or a combination of carbon and heteroatoms, i.e.,heterocycles such as a pyrimidine or quinazoline. Although smallmolecules can have any molecular weight, they generally includemolecules that are less than about 5,000 daltons. For example, suchsmall molecules can be less than about 1000 daltons and, preferably, areless than about 750 daltons or, more preferably, are less than about 500daltons. Small molecules and other non-peptidic Cadherin-11 antagonistscan be found in nature (e.g., identified, isolated, purified) and/orproduced synthetically (e.g., by traditional organic synthesis,bio-mediated synthesis, or a combination thereof). See e.g. Ganesan,Drug Discov. Today 7(1): 47-55 (January 2002); Lou, Drug Discov. Today,6(24): 1288-1294 (December 2001). Examples of naturally occurring smallmolecules include, but are not limited to, hormones, neurotransmitters,nucleotides, amino acids, sugars, lipids, and their derivatives.

A small molecule Cadherin-11 antagonist according to the presentinvention, and physiologically acceptable salts thereof, can inhibit thehomotypic binding of a Cadherin-11 protein (e.g., by directly competingwith a donor sequence in the EC1 domain of a Cad-11 protein for bindingto the binding pocket of another Cadherin-11, by directly competing withthe binding pocket in the EC1 domain of a Cad-11 protein for binding toa donor sequence of another Cadherin-11).

Nucleic Acid Antagonists

Cad-11 antagonists of the invention can also be nucleic acid molecules(e.g., oligonucleotides) that bind to the EC1 domain of a humanCadherin-11. Suitable nucleic acid Cad-11 antagonists include aptamers,which are capable of binding to a particular molecule of interest (e.g.,the EC1 domain of human Cadherin-11) with high affinity and specificitythrough interactions other than classic Watson-Crick base pairing (Tuerkand Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818(1990)).

Aptamers, like peptides generated by phage display or monoclonalantibodies (MAbs), are capable of specifically binding to selectedtargets and, through binding, block their targets' ability to function.Created by an in vitro selection process from pools of random sequenceoligonucleotides, aptamers have been generated for over 100 proteinsincluding growth factors, transcription factors, enzymes,immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size(30-45 nucleotides), binds its target with sub-nanomolar affinity, anddiscriminates against closely related targets (e.g., will typically notbind other proteins from the same gene family). A series of structuralstudies have shown that aptamers are capable of using the same types ofbinding interactions (hydrogen bonding, electrostatic complementarity,hydrophobic contacts, steric exclusion, etc.) that drive affinity andspecificity in antibody-antigen complexes.

An aptamer that binds to a target of interest (e.g., an EC1 domain of ahuman Cad-11 protein) can be generated and identified using a standardprocess known as “Systematic Evolution of Ligands by ExponentialEnrichment” (SELEX), described in, e.g., U.S. Pat. No. 5,475,096 andU.S. Pat. No. 5,270,163.

Identification of Cadherin-11 Antagonists

Agents having Cadherin-11 binding specificity, including smallmolecules, can be identified in a screen, for example, a high-throughputscreen of chemical compounds and/or libraries (e.g., chemical, peptide,nucleic acid libraries).

Antibodies that specifically bind the EC1 domain of human Cadherin-11can be identified, for example, by screening commercially availablecombinatorial antibody libraries (Dyax Corp., MorphoSys AG). Suitablecombinatorial antibody libraries and standard methods of screening theselibraries are described in Hoet et al., Nature Biotechnology23(3):344-348 (2005) and Rauchenberger et al., J. Biol. Chem.278(40):38194-38205 (2003), the contents of which are incorporatedherein by reference. Such libraries or collections of molecules can alsobe prepared using well-known chemical methods.

Alternatively murine antibodies that specifically bind the EC1 domain ofhuman Cadherin-11 can be identified, for example, by immunizing micewith EC1 protein domains or EC1 peptides along with an adjuvant to breaktolerance to the antigen. These antibodies can be screened for thedesired specificity and activity and then humanized using knowntechniques to create suitable agents for the treatment of human disease.

Compounds or small molecules can be identified from numerous availablelibraries of chemical compounds from, for example, the ChemicalRepository of the National Cancer Institute and the Molecular LibrariesSmall Molecules Repository (PubChem), as well as libraries of theInstitute of Chemistry and Cell Biology at Harvard University and otherlibraries that are available from commercial sources (e.g., Chembridge,Peakdale, CEREP, MayBridge, Bionet). Such libraries or collections ofmolecules can also be prepared using well-known chemical methods, suchas well-known methods of combinatorial chemistry. The libraries can bescreened to identify compounds that bind and inhibit Cadherin-11.

Identified compounds can serve as lead compounds for furtherdiversification using well-known methods of medicinal chemistry. Forexample, a collection of compounds that are structural variants of thelead can be prepared and screened for Cadherin-11 binding and/orinhibitory activity. This can result in the development of a structureactivity relationship that links the structure of the compounds tobiological activity. Compounds that have suitable binding and inhibitoryactivity can be developed further for in vivo use.

Agents that bind Cadherin-11 can be evaluated further for Cadherin-11antagonist activity. For example, a composition comprising a Cadherin-11protein can be used in a screen or binding assay to detect and/oridentify agents that bind and antagonize the Cadherin-11 protein.Compositions suitable for use include, for example, cells that naturallyexpress a Cadherin-11 protein (e.g., a synoviocyte), extracts of suchcells, and recombinant Cadherin-11 protein.

An agent that binds a Cadherin-11 protein can be identified in acompetitive binding assay, for example, in which the ability of a testagent to inhibit the binding of Cadherin-11 to a reference agent isassessed. The reference agent can be a full-length Cad-11 protein or aportion thereof that comprises the EC1 domain. The reference agent canbe labeled with a suitable label (e.g., radioisotope, epitope label,affinity label (e.g., biotin and avidin or streptavadin), spin label,enzyme, fluorescent group, chemiluminescent group, dye, metal (e.g.,gold, silver), magnetic bead) and the amount of labeled reference agentrequired to saturate the Cadherin-11 protein in the assay can bedetermined. The specificity of the formation of the complex between theCadherin-11 protein and the test agent can be determined using asuitable control (e.g., unlabeled agent, label alone).

The capacity of a test agent to inhibit formation of a complex betweenthe reference agent and a Cadherin-11 protein can be determined as theconcentration of test agent required for 50% inhibition (IC₅₀ value) ofspecific binding of labeled reference agent. Specific binding ispreferably defined as the total binding (e.g., total label in complex)minus the non-specific binding. Non-specific binding is preferablydefined as the amount of label still detected in complexes formed in thepresence of excess unlabeled reference agent. Reference agents suitablefor use in the method include molecules and compounds which specificallybind to Cadherin-11, e.g., an antibody that binds Cadherin-11.

An agent that antagonizes a Cadherin-11 protein can be identified byscreening for agents that have an ability to antagonize (reduce,prevent, inhibit) one or more activities of Cadherin-11, such as, forexample, a binding activity (e.g., homotypic Cad-11 binding). Suchactivities can be assessed using an appropriate in vitro or in vivoassay. Exemplary assays for Cadherin-11 activity have been describedpreviously (Patel, S D, et al., Cell 124: 1255-1268 (2006); Lee et al.,Science 315:1006-1010 (2007)).

Once a Cadherin-11 antagonist is identified, the ability of theCadherin-11 antagonist to interfere with (e.g., reduce, inhibit,prevent) one or more biological functions or properties associated withCadherin-11 activity in a cell can be assessed, for example, using acell-based assay designed to measure a particular biological function orproperty associated with Cadherin-11. Biological functions andproperties that are known to be associated with Cadherin-11 expressionand/or activity include, but are not limited to, cell adhesion, cellmigration, cell invasion, cell sorting, cell condensation, cellrearrangement, maintenance of tissue integrity and architecture, contactinhibition of cell proliferation and malignant transformation of cancer(e.g., tumor) cells (Kiener and Brenner, Arthritis Res Ther. 7(2):49-54(2005)). In addition Cad-11 antagonists are shown herein to inhibitproduction of active MMPs by synoviocytes. Suitable assays for assessingone or more biological functions of cadherins are known to those ofskill in the art (see, e.g., Patel, S D, et al., Cell 124: 1255-1268(2006)) and include, for example, the cell aggregation assay describedherein (see Exemplification, Materials and Methods section).

Methods of Therapy

Without wishing to be bound by any one theory, it is believed that thefirst about 35 amino acids (e.g., about 33 to about 37 amino acids) ofthe EC1 domain of Cad-11 are required for homotypic cadherin binding andthat agents that specifically bind to this region of Cad-11 caneffectively inhibit binding between Cad-11 molecules. Accordingly, suchagents are useful in the treatment and prevention of inflammatory jointdisorders (e.g., rheumatoid arthritis) associated with Cad-11 expressionand activity in synoviocytes and other cell types in inflamed joints.Thus, one aspect of the present invention relates to a method fortreating an inflammatory joint disorder in a mammalian subjectcomprising administering to the subject a therapeutically effectiveamount of a Cadherin-11 antagonist that binds a human Cadherin-11 EC1domain peptide (SEQ ID NO:3).

Using the methods of the invention, an inflammatory joint disorder in amammal (e.g., a human) can be treated by administering a Cadherin-11antagonist of the invention (e.g., antibodies, fusion proteins, smallmolecules, nucleic acids, peptides, peptidomimetics) in an amount thatis sufficient to provide a therapeutic benefit, for example, byinhibiting the aggregation of cells, or inhibiting the migration ofcells, or inhibiting expression of active proteases or inflammatorymolecules by cells, that express Cadherin-11 in an articulated joint(e.g., synoviocytes).

Accordingly, one aspect of the invention relates to a method fortreating an inflammatory joint disorder in a mammalian subjectcomprising administering to the subject a therapeutically effectiveamount of a Cadherin-11 antagonist of the invention. The inflammatoryjoint disorder can be any disorder that is associated with orcharacterized by Cadherin-11 expression in cells (e.g., synoviocytes) ofan articulated joint. Examples of inflammatory joint disorders that canbe treated by the present invention include, but are not limited to,rheumatoid arthritis, psoriatic arthritis, Reiter's syndrome andankylosing spondylitis. In a particular embodiment, the inflammatoryjoint disorder is rheumatoid arthritis.

In one aspect, a therapeutically effective amount of a Cadherin-11antagonist is administered to a patient in need thereof. The amount ofthe Cadherin-11 antagonist to be administered (e.g., a therapeuticallyeffective amount) can be determined by a clinician using the guidanceprovided herein and other methods known in the art and is dependent onseveral factors including, for example, the particular agent chosen, thesubject's age, sensitivity, tolerance to drugs and overall well-being.For example, suitable dosages for Cad-11 antagonists that are antibodiescan be from about 0.01 mg/kg to about 300 mg/kg body weight pertreatment and preferably from about 0.01 mg/kg to about 100 mg/kg, fromabout 0.01 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 10 mg/kgbody weight per treatment. Suitable dosages for a small molecule Cad-11antagonist can be from about 0.001 mg/kg to about 100 mg/kg, from about0.01 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 10 mg/kg,from about 0.01 mg/kg to about 1 mg/kg body weight per treatment.Suitable dosages for Cadherin-11 antagonists that are proteins orpeptides (linear, cyclic, mimetic), will result in a plasmaconcentration of the peptide from about 0.1 μg/mL to about 200 μg/mL.Determining the dosage for a particular agent, patient and cancer iswell within the abilities of one skilled in the art. Preferably, thedosage does not cause, or produces minimal, adverse side effects (e.g.,immunogenic response, nausea, dizziness, gastric upset, hyperviscositysyndromes, congestive heart failure, stroke, pulmonary edema).

A therapeutically effective amount of a Cadherin-11 antagonist can beadministered alone, or in combination with one or more other therapeuticagents (e.g., anti-inflammatory agents). Suitable anti-inflammatoryagents that are useful for treating inflammatory joint disorders,particularly RA, which can be administered in combination with Cad-11antagonists of the invention, include, but are not limited to, (i)non-steroidal anti-inflammatory drugs (NSAIDs; e.g., detoprofen,diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen,indomethacin, ketoprofen, meclofenameate, mefenamic acid, meloxicam,nabumeone, naproxen sodium, oxaprozin, piroxicam, sulindac, tolmetin,celecoxib, rofecoxib, aspirin, choline salicylate, salsalte, and sodiumand magnesium salicylate); (ii) steroids (e.g., cortisone,dexamethasone, hydrocortisone, methylprednisolone, prednisolone,prednisone, triamcinolone); (iii) DMARDs, i.e., disease modifyingantirheumatic drugs (e.g., cyclosporine, azathioprine, methotrexate,leflunomide, cyclophosphamide, hydroxychloroquine, sulfasalazine,D-penicillamine, minocycline, and gold); or (iv) recombinant proteins(e.g., ENBREL® (etanercept, a soluble TNF receptor), REMICADE®(infliximab, a chimeric monoclonal anti-TNF antibody), ORENCIA®(abatabacept, a soluble CTLA4 receptor), ACTEMRA® (Tocilizumab, amonoclonal antibody to the IL-6 receptor), and RITUXAN® (rituximab, amonoclonal antibody to CD20).

Thus, a Cadherin-11 antagonist can be administered as part of acombination therapy (e.g., with one or more other therapeutic agents).The Cad-11 antagonist can be administered before, after or concurrentlywith one or more other therapeutic agents. In some embodiments, theCadherin-11 antagonist and other therapeutic agent can beco-administered simultaneously (e.g., concurrently) as either separateformulations or as a joint formulation. Alternatively, the agents can beadministered sequentially, as separate compositions, within anappropriate time frame as determined by the skilled clinician (e.g., atime sufficient to allow an overlap of the pharmaceutical effects of thetherapies). The Cadherin-11 antagonist and one or more other therapeuticagents can be administered in a single dose or in multiple doses, in anorder and on a schedule suitable to achieve a desired therapeutic effect(e.g., a reduction in and/or inhibition of joint inflammation). Suitabledosages and regimens of administration can be determined by a clinicianand are dependent on the agent(s) chosen, pharmaceutical formulation androute of administration, various patient factors and otherconsiderations.

The effectiveness of a therapy (e.g., the reduction or elimination ofjoint inflammation and/or the prevention or inhibition of jointinflammation) can be determined by any suitable method (e.g., imaging(MRI, NMR)).

According to the methods of the invention, a therapeutically effectiveamount of a Cad-11 antagonist is administered to a mammalian subject totreat an inflammatory joint disorder. The term “mammalian subject” isdefined herein to include mammals such as primates (e.g., humans) cows,sheep, goats, horses, dogs cats, rabbits, guinea pigs, rats, mice orother bovine, ovine, equine, canine feline, rodent and murine species.

Agents that are Cad-11 antagonists can be administered to a mammaliansubject by a variety of routes. For example, the agent can beadministered by any suitable parenteral or nonparenteral route,including, for example, topically (e.g., cream, ointment), or nasally(e.g., solution, suspension). Parenteral administration can include, forexample, intraarticular, intramuscular, intravenous, intraventricular,intraarterial, intrathecal, subcutaneous, or intraperitonealadministration. The agent can also be administered orally (e.g., incapsules, suspensions, tablets or dietary), transdermally,intradermally, topically, by inhalation (e.g., intrabronchial,intranasal, oral inhalation or intranasal drops), transmucosally orrectally. Administration can be local or systemic as appropriate, andmore than one route can be used concurrently, if desired. Localizedadministration of a Cad-11 antagonist can be achieved by intraarticularinjection (e.g., direct injection of the agent into a joint). Thepreferred mode of administration can vary depending upon the particularagent chosen. However, systemic intravenous or subcutaneousadministration is generally preferred for antibodies.

Delivery can also be by injection into the brain or body cavity of apatient or by use of a timed release or sustained release matrixdelivery systems, or by onsite delivery using micelles, gels andliposomes. Nebulizing devices, powder inhalers, and aerosolizedsolutions are representative of methods that may be used to administersuch preparations to the respiratory tract. Delivery can be in vitro, invivo, or ex vivo.

Agents that are proteins (e.g., fusion protein) can be administered viain vivo expression of recombinant protein. In vivo expression can beaccomplished by somatic cell expression according to suitable methods(see, e.g., U.S. Pat. No. 5,399,346). Further, a nucleic acid encodingthe protein can also be incorporated into retroviral, adenoviral orother suitable vectors (preferably, a replication deficient infectiousvector) for delivery, or can be introduced into a transfected ortransformed host cell capable of expressing the protein for delivery. Inthe latter embodiment, the cells can be implanted (alone or in a barrierdevice), injected or otherwise introduced in an amount effective toexpress the protein in a therapeutically effective amount.

Nucleic acid-based Cadherin-11 antagonists (e.g., aptamers) can beintroduced into a mammalian subject of interest in a number of ways. Forinstance, nucleic acids may be expressed endogenously from expressionvectors or PCR products in host cells or packaged into synthetic orengineered compositions (e.g., liposomes, polymers, nanoparticles) thatcan then be introduced directly into the bloodstream of a mammaliansubject (by, e.g., injection, infusion). Anti-Cadherin-11 nucleic acidsor nucleic acid expression vectors (e.g., retroviral, adenoviral,adeno-associated and herpes simplex viral vectors, engineered vectors,non-viral-mediated vectors) can also be introduced into a mammaliansubject directly using established gene therapy strategies and protocols(see e.g., Tochilin V. P. Annu Rev Biomed Eng 8:343-375, 2006;Recombinant DNA and Gene Transfer, Office of Biotechnology Activities,National Institutes of Health Guidelines).

Agents that are Cadherin-11 antagonists (e.g., small molecules) can beadministered to a mammalian subject as part of a pharmaceutical orphysiological composition, for example, as part of a pharmaceuticalcomposition comprising a Cadherin-11 antagonist and a pharmaceuticallyacceptable carrier. Formulations or compositions comprising aCadherin-11 antagonist or compositions comprising a Cadherin-11antagonist and one or more other therapeutic agents (e.g., ananti-inflammatory agent) will vary according to the route ofadministration selected (e.g., solution, emulsion or capsule). Suitablepharmaceutical carriers can contain inert ingredients which do notinteract with the Cadherin-11 antagonist. Standard pharmaceuticalformulation techniques can be employed, such as those described inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa. Suitable pharmaceutical carriers for parenteral administrationinclude, for example, sterile water, physiological saline,bacteriostatic saline (saline containing about 0.9% mg/ml benzylalcohol), phosphate-buffered saline, Hank's solution, Ringer's lactateand the like. Formulations can also include small amounts of substancesthat enhance the effectiveness of the active ingredient (e.g.,emulsifying agents, solubilizing agents, pH buffering agents, wettingagents). Methods of encapsulation compositions (such as in a coating ofhard gelatin or cyclodextran) are known in the art. For inhalation, theagent can be solubilized and loaded into a suitable dispenser foradministration (e.g., an atomizer or nebulizer or pressurized aerosoldispenser).

The pharmaceutical agent can be administered as a neutral compound or asa salt or ester. Pharmaceutically acceptable salts include those formedwith free amino groups such as those derived from hydrochloric,phosphoric, acetic, oxalic or tartaric acids, and those formed with freecarboxyl groups such as those derived from sodium, potassium, ammonium,calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. Salts of compounds containing anamine or other basic group can be obtained, for example, by reactingwith a suitable organic or inorganic acid, such as hydrogen chloride,hydrogen bromide, acetic acid, perchloric acid and the like. Compoundswith a quaternary ammonium group also contain a counteranion such aschloride, bromide, iodide, acetate, perchlorate and the like. Salts ofcompounds containing a carboxylic acid or other acidic functional groupcan be prepared by reacting with a suitable base, for example, ahydroxide base. Salts of acidic functional groups contain acountercation such as sodium or potassium.

The present invention will now be illustrated by the following Examples,which are not intended to be limiting in any way.

EXEMPLIFICATION Example 1 Identification of Fabs Having BindingSpecificity for an Epitope within the N-Terminal 35 Amino Acids of theEC1 Domain of Human Cadherin-11 Materials and Methods Western Blotting

Proteins were separated by SDS-PAGE and transferred to a nitrocellulose(NC) membrane using standard methods. Briefly NC membrane was rinsedwith tris buffered-saline-tween (TBST) (8.8 g/L of NaCl, 0.2 g/L of KCl,3 g/L of Tris base, 500 ul/L of Tween-20, pH to 7.4). The membrane wasblocked with 4% BSA dissolved in TBST for hour at 22° C. The NC membranewas rinsed 3× for 5 min each with TBST. Mouse anti-human Cad-11 antibodywas diluted to 0.5 μg/ml in TBST and the NC was incubated for 1 hour at22° C. The NC membrane was rinsed 3× for 5 minutes each in TBST. Goatanti-mouse Ig antibody conjugated with horse radish peroxidase (HRP) wasdiluted to 1 μg/ml in TBST and the NC membrane was incubated insecondary solution for a minimum time of 1 hour @ room temperature (RT)at 22° C. The NC membrane was rinsed 3× for 5 min each in TBST. Signalwas developed using standard HRP method.

ELISA

The antigen (either 5 μg/ml or 50 μg of Cad-11-EC-1-Fc or 5 μg/ml ofCadherin peptide) was diluted in a buffer and used to coat the platesovernight at 4° C. The plates were washed and then blocked with 1.5%BSA, 5% low fat milk powder in PBS Dilution buffer: 1.5% BSA, 2.5% lowfat milk powder, 0.1% Tween-20 in PBS. The plates were then incubatedwith bacterial lysate containing the anti-Cad-11 human fAbs or purifiedanti-Cad-11 human fAbs for 1 hr. After washing, the secondary antibody(Cy5-conjugated a-hu-Fab diluted 1/100) was applied for 25 min. Theplates were then washed and the resulting fluorescence read.

Results

Three sets of previously reported Cadherin-11-specific antibodies weretested for an ability to bind to the EC1 domain of human Cadherin-11.These antibodies included antibodies that were raised against a mouseCadherin-11-Fc fusion protein immunogen in Cadherin-11 knock-out ordeficient mice (Lee et al., Science 315:1006-1010 (2007)), antibodiesthat were raised in Cadherin-11 wild type mice against a humanCadherin-11-Fc fusion protein immunogen that had been produced in CHOcells (Valencia et al., J. Exp. Med. 200(12):1673-1679 (2004)), andantibodies that were raised in Cadherin-11 wild type mice against abacterially-produced protein containing the EC1-3 domains of humanCadherin-11. These antibodies were tested by western analysis for anability to bind fusion proteins that contained only the EC1 domain ofhuman Cad-11 (Cadherin-11-EC1-Fc), the EC1 and EC2 domains of Cad-11(Cad11-EC1/2-Fc) or all 5 EC domains of Cad-11 (Cadherin-11-EC1-5-Fc).None of the antibodies tested recognized the EC1-Fc or the EC1-2-Fcfusion proteins on a Western blot (FIG. 1A). However, antibodies fromeach of the three sets tested recognized the human Cad-11-Fc fusionprotein that included extracellular domains 1 through 5 (FIG. 1B). Theseresults indicate that the tested antibodies did not bind to the EC1 orEC2 domains of human Cad-11, but recognized epitopes elsewhere in theextracellular region of this protein.

The available published anti-Cad11 antibodies that bind Cad11 expressingcells, 13C2, 23C6, 5F82 (Lifespan Science) and 283416 (R&D Systems), aswell as the Cad11 EC1-binding antibody H1M1, and the control antibody,MOPC, were tested by ELISA for the ability to bind fusion proteins thatcontained only the EC1 domain of human Cad-11 (Cadherin-11-EC1-Fc) orall 5 EC domains of Cad-11 (Cadherin-11-EC1-5-Fc). None of the availablepublished anti-Cad11 antibodies tested recognized the EC1-Fc (FIG. 1B,open bars) (data for 283416 is not shown here). However, the Cad11 EC1binding H1M1 antibody bound both the Cadherin-11-EC1-Fc andCadherin-11-EC1-5-Fc (FIG. 1B closed bar). The control MOPC antibodybound neither fusion protein. These results indicate that the availablepublished anti-Cad11 antibodies do not bind to the EC1 domain of humanCad-11, but recognized epitopes elsewhere in the extracellular region ofthis protein.

To create an antibody specific for an epitope within the N-terminal 35amino acids of the EC1 domain of human Cadherin-11, a phage displaylibrary (MorphoSys AG) encoding human Fabs was screened. Candidate Fabswere identified using two selection criteria—a positive selection forbinding to a peptide that included the first 35 amino acids of the humanCadherin-11 EC1 domain, and a negative selection for binding tocorresponding peptides from the EC1 domains of two closely related andhighly homologous cadherins, Cadherin-8 and MN-Cadherin (FIG. 2). ELISAwas used to assess binding.

Two screens were conducted. In the first screen, 96 Fab clones thatbound the Cadherin-11 EC1 peptide were identified by ELISA. Seven (7)candidate Fabs bound the Cad-11 EC-1 peptide; however, only two of thesebound to both EC-1 peptide and the EC1-2-Fc fusion protein. One of thesetwo Fabs also bound to MN-Cad peptide. Accordingly, only one of theseven Fab clones specifically bound the EC1-Fc fusion protein, but didnot bind to both MN-Cad and Cadherin-8 EC1 domain peptides. In a secondscreen, similar results were observed, as only 1 of 96 Fabs (clone F9)showing specificity for the Cadherin-11 EC1 peptide and EC1-2-Fc fusionprotein, failed to bind MN-Cad and Cadherin-8 EC1 domain peptides (FIG.3). The majority of the Cad-11 EC1 domain-binding Fabs tested showedcross reactivity with the MN-Cad peptide, which contains an EEY CARsequence that overlaps with the EEY CAR sequence of Cad-11.

Example 2 A Fab that Binds the EC1 Domain of Cadherin-11 Inhibits Cad-11Mediated Cell Aggregation in an In Vitro Assay Materials and Methods InVitro Cadherin-11 Aggregation Assay

431-D cells grow in suspension and do not normally express any cadherinsand do not aggregate. 431-D-11 cells have been genetically modified toexpress Cad-11.

When 431-D-11 cells are incubated in media alone and they begin toaggregate over 40 min and the clumps of cells settle to the bottom ofwell and the remaining non-aggregated cells in suspension can bemeasured and the percentage of aggregated 431-D-11 calculated. For theaggregation assay, 431-D-11 cells (D-11 cells) were grown tosub-confluence in a flask and then were removed from the flask using0.05% Trypsin plus 0.53 mM EDTA. Approximately 2×10⁶ 431-D-11 cells wereadded to 2 ml of SME media (Dulbecco's Modified Eagle's Medium-highglucose, 0.1M Hepes pH 7.4 and 5 U/ml DNAse) and were preincubated for15 min on ice, either in the absence or presence of a test agent (e.g.,antibody, Fab, fusion protein). After pre-incubation with the testagent, the cells were transferred to a round bottom well on a 24-wellplate and incubated at 37° C. while rotating at 130 rpm on a rotaryshaker. As cells aggregate they sink to the bottom of the well. At 0 minand 40 min, 200 μl from the middle of the sample were removed from thewell and mixed with 25 μl of 8% glutaraldehyde to fix the cells. 200 μlof the fixed sample of cells were added to 9.8 ml of Coulter Counterisotonic saline solution and counted using a Coulter Counter set at the8 μm to 24 μm threshold. 3 cell counts per sample were recorded. Thepercentage of cell decrease or aggregated cells at 40 min. compared tothe percentage at the 0 min. time point was calculated.

Results

A candidate Fab (clone F9) having binding specificity for an epitopewithin the N-terminal 35 amino acids of the Cadherin-11 EC1 domain,which does not bind the EC1 domains of MN-Cad or Cad-8, was tested foran ability to inhibit Cad-11 mediated cell aggregation using an in vitroCadherin-11 cell aggregation assay. The candidate Fab significantlyinhibited Cadherin-11 mediated aggregation of cells at concentrations of1 μg/ml or lower (FIGS. 4 and 5). In contrast, a Fab made from the 13C2antibody that binds to an epitope in the extracellular region of Cad-11outside the EC1/2 domains inhibited Cadherin-11 aggregation only at aconcentration of 10 μg/ml, suggesting that the F9 Fab inhibits Cad-11activity more effectively at lower concentrations than antibodies whichbind to other portions of the extracellular domain of Cad-11.

Cad-11 mediated cell aggregation was also inhibited by variousanti-Cadherin-11-EC1 domain Fabs that were specific for either Cad-11alone, Cad-11 and Cad-8, Cad-11 and MN-Cad, or Cad-11, Cad-8 and MN-Cad(FIGS. 6 and 7). All cadherin-EC1-domain specific Fabs that were testedinhibited cell aggregation in vitro relative to the control samples(e.g., SME medium (FIG. 6), a Fab specific for GFP (FIG. 7).

Example 3 Generation of Cadherin-11/Immunoglobulin Fusion ProteinsContaining the EC1 Domain of Human Cadherin-11

The Cadherin-11 EC1 region was prepared from a vector encoding the fulllength human Cadherin-11 cDNA (human Cad-11 cloned into the Not1 andKpn-1 sites of the Invitrogen pCEP4® vector) using polymerase chainreaction (PCR) performed under standard conditions using the followingoligonucleotide primers to introduce EcoR1 and BglII sites (seeunderlined sequences in forward and reverse primers, respectively) intothe amplified product:

Forward Primer: (SEQ ID NO: 8) tttttttttgaattcatgaaggagaactactgtttacaagc           EcoRI Reverse Primer: (SEQ ID NO: 9)tttttttttagatctctggaccttgacaatgaattccgacgg           BglII

The amplified product was digested with restriction enzymes EcoR1 andBglII, and the digestion product was isolated and ligated into thepFUSE-hIgG2el-Fc1 vector (InvivoGen) using the corresponding EcoR1 andBglII sites. TOP10 competent bacteria (Invitrogen) were transformed asdescribed by the manufacturer with the ligation product and bacteriawith the Cadherin-11-EC1-Fc plasmid were selected with zeomycin.Cadherin-11-EC1-Fc plasmid was isolated, sequenced and then used totransiently transfect 293F cells. Conditioned media was collected andthe Cadherin-11-EC1-Fc fusion protein (SEQ ID NO:9) was purified usingtangential flow filtration followed by isolation on a 50/50 mix proteinA/protein G column equilibrated in 20 mM HEPES pH 7.4, 137 mM NaCl, 3 mMKCl and 1 mM CaCl2.

The purified Cadherin-11-EC1-Fc fusion protein was eluted from thecolumn using 0.1 M Glycine (pH 3) and

1 mM CaCl2 and into tubes containing 200 μl of 1M Tris pH 7.4, and 1 mMCaCl₂. The eluates with the Cad-11 fusion protein were then dialyzedagainst 20 mM Hepes (pH 7.4), 137 mM NaCl, 3 mM KCl and 1 mM CaCl₂. Thesize of the protein was confirmed by SDS PAGE (FIG. 10) and identity wasconfirmed by Western analysis using an antibody that recognizes thehuman Fc region (FIG. 11) and N-terminal sequencing (not shown).Cad-11-EC1-2-Fc was produced using techniques and conditions similar

those described above.

Example 4 A Cad-11-EC1-Fc Immunoglobulin Fusion Protein Inhibits Cad-11Mediated Cell Aggregation in an In Vitro Assay Materials and Methods

Cell Invasion/Migration into a Matrigel Plug

FLS migratory activity was assessed in Matrigel ECM-coated transwells inFLS media (Dulbecco's Modified Eagle's Medium-high glucose [Sigma#D7777], 10% Fetal Bovine Serum [Benchmark #100-106], 1%Pencillin-Streptomycin [Gibco 315140-122], 1% L-Glutamine [Gibco#25030], 0.5% Gentamicin [Gibco #15710-064]. Human FLS cell suspensionsin FLS medium containing 1×10⁴ cells were added to the control insert ormatrigel coated insert set in the well of a 24-well plate containing0.750 mL of

FLS medium. The plates were then incubated in a humidified tissueculture incubator at37° C., 5% CO₂ atmosphere for 22 hours.

To calculate the number of cells that migrated, non-invading cells wereremoved from the upper surface of the membrane of control inserts bywiping with a cotton swab. A second wipe using a cotton swap wetted withFLS medium is repeated. Control inserts were then fixed and stainedusing a differential staining kit [Fisher #122-911]. Inserts are allowedto dry and cells are counted in 4 quadrants of the control insert usinga microscope with a 10× objective. Triplicate inserts are counted andthe totals averaged.

To calculate the number of cells that invaded the matrigel inserts,non-invading cells were gently removed from the surface of the matrigelinsert by wiping with a cotton swab. A second wipe using a cotton swapwetted with FLS medium is repeated. Inserts were then fixed and stainedusing a differential staining kit [Fisher #122-911]. Inserts are allowedto dry and cells are counted in 4 quadrants of the control insert usinga microscope with a 10× objective. Triplicate inserts are counted andthe totals averaged.

Results

Cad-11-EC1-Fc significantly inhibited cell aggregation at aconcentration of 3 μg/ml, while the full length Cad-11-EC1-5-Fc proteincontaining all 5 EC domains of human Cad-11 inhibited Cad-11 mediatedaggregation at a concentration of 100 μg/ml (FIG. 13). These data showthat the Cad-11-EC1-Fc immunoglobulin fusion protein effectivelyinhibits Cad-11 mediated cell aggregation in an in vitro assay.

In addition, the ability of the Cad-11-EC1-Fc immunoglobulin fusionprotein to inhibit the invasion of human fibroblast like synoviocytes(FLS) into a matrigel plug was tested in vitro. Invasion of the FLS intomatrigel is a complex process that involves the expression of MMP1,MMP-3, MMP-13, serine proteases, and other proteins by the FLS todegrade the matrigel as well as the migration of the FLS into matrigel.In a separate assay we saw no inhibition of migration of FLS through anormal fiber insert. This suggests the impact of the EC-Fc or 13C2 mAbis to inhibit the degradation of the matrigel (a surrogate for jointcartilage). Both the Cad-11-EC1-Fc and murine anti-Cad-11 mAb 13C2inhibited FLS invasion into a matrigel plug in two independentexperiments.

Example 5 Generation of Antibodies Against an EC1 Domain Peptide ofHuman Cadherin-11 Materials and Methods

Balb/c mice were immunized bi-weekly in the foot pad nine times over aone month period with 0.01 mg of a peptide corresponding to the first 33amino acids of the human Cad-11 EC1 domain (GWVWN QFFVI EEYTG PDPVLVGRLH SDIDS GDG (SEQ ID NO:10)), covalently linked to BSA. This peptideis referred to herein as Peptide 4. Spleens from the immunized mice wereharvested and fused with a murine fusion partner, P3X63-Ag8.653, tocreate antibody-producing hybridomas. The hybridomas were expanded andsubcloned at either 10, 3 or 0.5 cells/well, and the anti-Cad-11antibody-containing media from the hybridomas were screened in an ELISAfor the ability to specifically bind Cad-11 EC1-2 domain-containingprotein produced in bacteria. Anti-Cad-11 antibody-containing media fromthese Peptide 4 hybridomas were screened concurrently for the absence ofbinding to proteins encompassing the EC1-2 domains of human Cad-8 andMN-Cadherin. 96-well EIA plates were coated overnight at 4° C. with 0.05ml of 0.0 to 0.3 mg/ml of each of the EC1-2 Cad proteins and then washedseveral times with saline buffer. Plates were then blocked with 0.25 mlof casein-PBS buffer and washed several times with saline buffer.Hybridoma media containing the anti-Cad-11 antibody were incubated neatin each well for 1 hr at 22° C. and then washed twice with PBS-Tween(0.05%). 100 μl of a 1/1000 dilution of a goat anti-mouse IgG secondaryantibody were added to each well, incubated for 30 min at 22° C., andthen washed twice with PBS-Tween (0.05%). 100 μl/well of roomtemperature TMB (3,3′,5,5′-tetramethylbenzidine) reagent was added toeach well and color was allowed to develop for 5 min at 22° C. Thereaction was stopped with 100 μl of room temperature 2N sulfuric acidand the plate was read at 450 nm on a Wallac 1420 microplate reader.

The specificity of H1M1 and H14 anti-Cad-11 antibodies was testedfurther using an ELISA against the first 33 amino acids of the EC1domains of Cad-11, Cad-7, Cad-8, Cad-20, Cad-24, Cad-9, Cad-18, andMN-Cad. Peptides corresponding to the region of Cad-7, Cad-8, Cad-20,Cad-24, Cad-9, Cad-18, MN-Cad that overlapped with the G1-G33 region ofthe Cad-11 EC1 domain were synthesized and conjugated to biotin. 100 μlof a 30 ng/ml solution of each of these peptides in PBS-Tween (0.05%)were incubated in each well of a 96-well Netravidin plate for 2-3 hrs at4° C. and then washed twice with PBS-Tween (0.05%). Variousconcentrations of the anti-Cad-11 antibody were incubated in each wellfor 1 hr at 22° C. and then washed twice with PBS-Tween (0.05%). 100 μlof a 1/1000 dilution of a goat anti-mouse IgG secondary antibody wereadded to each well, incubated for 30 min at 22° C., and then washedtwice with PBS-Tween (0.05%). 100 μl/well of room temperature TMB(3,3′,5,5′-tetramethylbenzidine) reagent was added to each well andcolor was allowed to develop for 5 min at 22° C. The reaction wasstopped with 100 μl of room temperature 2N sulfuric acid and the platewas read at 450 nm on a Wallac 1420 microplate reader.

Media from wells containing positive anti-Cad-11 antibody hybridomaswere tested for the ability to bind to Cad-11 expressing cells. FrozenCad-11-expressing 431D cells were thawed and washed twice in HanksBalanced Saline Solution (HBSS) containing Ca²⁺ (0.137 M NaCl, 5.4 mM,KCl 0.25, mM Na₂HPO₄, 0.44 mM KH₂PO₄, 1.3 mM CaCl₂, 1.0 mM MgSO₄ and 4.2mM NaHCO) and then resuspended at 10⁶ cells/ml in HBSS containing Ca²⁺.10⁵ cells/well were stained with either a 50% or 16% anti-Cad-11antibody media for 45 min on ice, washed twice in HBSS containing Ca²⁺,stained with a secondary goat anti-mouse IgG antibody conjugated withphytoerytherin (Jackson ImmunoResearch, West Grove, Pa.) a concentrationof 1% for 45 min on ice and then washed again twice in HBSS containingCa²⁺. Cells were then resuspended in 400 μl of HBSS containing Ca²⁺ and1% formaldehyde and subsequently analyzed on a FACScalibur (BectonDickenson, Franklin Lakes, N.J.) for PE positive cells.

Results

Anti-Cad-11 antibody-containing media from the Peptide 4 hybridomasbound to the Cad-11 EC1-2 protein (FIG. 16, HL vs CAD11), but notproteins containing the EC1-2 domains of Cad-8 and MN-Cad (FIG. 16, HLvs CAD8 and HL vs MNCAD, respectively). Control hybridoma media did notbind any of the cadherin proteins tested (FIG. 16, Media vs CAD11, Mediavs CAD8, and Media vs MNCAD). These data demonstrate the presence ofCad-11 specific antibodies to Peptide 4 in the hybridomas.

Two Peptide 4 hybridomas, referred to herein as H1M1 and H14, bound tocells expressing human Cad-11 protein (FIGS. 17A-C and 17G-I), but notto non-Cad-11 control 431-D cells (FIGS. 17D-17F). The hybridoma cellline referred to as H1M1 has the A.T.C.C. designation number ______,having been deposited on Jan. 8, 2009. The hybridoma cell line referredto as H14 has the A.T.C.C. designation number ______, having beendeposited on Jan. 9, 2009. These hybridomas contain anti-Cad-11antibodies that recognize both Peptide 4 and Cad-11-expressing cells invitro. The binding of these antibodies to Cad-11-expressing cells wasshown to titrate with the amount of Peptide 4 antibody that was used, asshown in the plots of the titration of H1M1 (FIG. 18A) and H14 (FIG.18B) versus the intensity of cell staining from the mean fluorescenceintensity (MFI).

The H1M1 and H14 Peptide 4 anti-Cad-11 antibodies demonstrated >100-foldhigher binding to Cad-11 than to any of the other cadherins tested,which included Cad-7, Cad-8, Cad-20, Cad-24, Cad-9, Cad-18, and MN-Cad.In most cases, no binding of H1M1 and H14 anti-Cad-11 antibodies to theother cadherins was observed. The anti-Cad-11 antibody H14 showed strongbinding to Cad-11 (FIG. 19A), with 468-fold lower binding to Cad-8 (FIG.19A), and virtually no binding to Cad-7, MN-Cad, Cad-9, Cad-18, Cad-20or Cad-24 (FIG. 19B). Similarly, the anti-Cad-11 antibody H1M1 showedstrong binding to Cad-11 (FIG. 20), with 365-fold lower binding to Cad-8(FIG. 20), and no binding to Cad-7, MN-Cad, Cad-9, Cad-18, Cad-20 orCad-24 (data not shown).

Example 6 The Anti-Cad-11 EC1 Domain Antibodies H1M1 and H14 BindEpitopes in the Cad-11 EC1 Domain that Include the Amino Acid SequenceGPDP Materials and Methods

To determine the epitope within the Cad-11 EC1 domain that the Peptide 4Cad-11 EC1 antibodies H1M1 and H14 bind, four different peptidesspanning the first 37 amino acids of the EC1 region (see FIG. 22) wereimmobilized in an ELISA format and the ability of the H1M1 and H14antibodies to bind each of the four peptides was determined. 96-wellReactibind plates were coated overnight at 4° C. with 0.3 ng/well ofPeptide 1 (amino acids G1-P18 of the Cad-11 EC1 domain), 0.3 ng/well ofPeptide 2 (amino acids G15-N34 of the Cad-11 EC1 domain), 0.3 ng/well ofPeptide 3 (amino acids V19-Y37 of the Cad-11 EC1 domain), 0.3 ng/well ofthe immunogen Peptide 4 (amino acids G1-G33 of the Cad-11 EC1 domain),20 ng of a fusion protein including the entire EC1 domain (EFL), or 20ng of control human Ig (Fc-block). The wells were washed twice withPBS-Tween (0.05%), blocked with casein in dH₂0 for 3 hrs at 22° C. andthen washed again twice with PBS-Tween (0.05%). Various dilutions of thedifferent Peptide 4 CAD-11 EC1 domain antibodies were transferred to thepeptide- or protein-coated wells, incubated for 45 min at 22° C. andthen washed twice with PBS-Tween (0.05%). 100 μl of a 1/1000 dilution ofgoat anti-mouse IgG secondary antibody (Jackson ImmunoResearch, WestGrove, Pa.) were added to each well, incubated for 30 min at 22° C. andthen washed twice with PBS-Tween (0.05%). 100 μl/well of roomtemperature TMB reagent was added to each well and color was allowed todevelop for 5 min at 22° C. The reaction was stopped with 100 μl of roomtemperature 2 N sulfuric acid and the plate was read at a wavelength of450 nm on a Wallac 1420 microplate reader.

Results

The Peptide 4 anti-Cad-11 antibodies H1M1 at 1:11 (FIG. 21A) and H14 at1:23 (FIG. 21B) both bound the Peptide 4 (PEP4) immunogen, as well asthe EC1 domain fusion protein (EFL), in the ELISA as indicated byelevated OD450 plate readings relative to the control. Neither of theseantibodies bound to the human IgG control (Fc block). In addition, bothantibodies bound Peptide 2 (PEP2), but not Peptide 1 (PEP1) or Peptide 3(PEP3), in the ELISA (FIGS. 21A and 21B).

These results suggest that the anti-Cad-11 EC1 domain antibodies H1M1and H14 bind a common epitope in Peptides 2 and 4 that is not present inthe overlapping Peptide 3. Amino acids shared by Peptides 2 and 4 thatare upstream of Peptide 3 are highlighted in the boxed region shown inFIG. 22. These four amino acids, GPDP (SEQ ID NO:11), beginning at G15of the Cad-11 EC1 domain, are likely part of the epitope recognized bythe H1M1 and H14 antibodies.

Example 7 The Anti-Cad-11 EC1 Domain Antibodies H1M1 and H14 InhibitAggregation of Cad-11-Expressing Cells In Vitro Materials and Methods

To assess the ability of the Cad-11 antibodies to inhibit Cad-11mediated cell aggregation, 30 μg/ml of the H1M1 Peptide 4 antibody wascultured with 75,000 Cad-11 expressing A-431-D epidermoid carcinomacells in 0.5 ml of DMEM-high glucose, 20 mM Hepes pH 7.4, 10% FCS and 10U/ml DNAse in a 24-well round bottom polypropylene plate. The 24-wellplates were placed on a rotating platform at approximately 60 rpm andincubated with 5% CO₂ overnight at 37° C. The next day, cell aggregationwas assessed after photographing the plates at 100× (for H1M1experiment) or 40× (for H14 experiment) magnification.

Results

In the presence of a control isotype antibody (30 μg/ml), theCad-11-expressing cells formed large masses (FIG. 23A), while theparental Cad-11 negative cells remain as single or double cell groups(FIG. 23C). The H1M1-treated Cad-11 cells remained as small clumps ofcells (FIG. 23B) that did not progress to form the large masses obtainedusing the control antibody.

Using the same assay, the anti-Cad-11 antibody H14 was also shown toinhibit Cad-11-mediated aggregation. While the parentalCad-11-expressing cells formed large clusters of aggregated cells (FIG.24A), the H14 antibody (FIG. 24B) inhibited aggregation at aconcentration of 30 μg/ml, as cell clusters were small and infrequent.These results indicate that the anti-Cad-11 antibodies H1M1 and H14inhibit Cad-11-mediated cell aggregation in vitro.

Example 8 The Anti-Cad-11 EC1 Domain Antibodies, H1M1 and H14, InhibitArthritis-Associated Joint Swelling In Vivo in a Murine Model ofRheumatoid Arthritis Materials and Methods

Study 1—Six-week-old male C57/B16 mice were injected with 150 μl of KBNsera on day 0 and day 2. KBN sera-treated mice received either salineinjections (FIG. 25, unfilled triangles) or were treated with differentdoses of the H1M1 anti-Cad-11 EC1 antibody. Treatment regimens includeddosing on day 0 with 0.5 mg of antibody/mouse and every second day (q2d)thereafter with 0.1 mg of antibody/mouse (0.5 mg+0.1 mg) (FIG. 25,filled triangles); dosing on day 0 with 0.5 mg of antibody/mouse (0.5mg) (FIG. 25, diamonds); dosing every second day (q2d) with 0.1 mg ofantibody/mouse (0.1 mg+0.1 mg) (FIG. 25, squares); or dosing everysecond day (q2d) with 0.3 mg of antibody/mouse (0.3 mg+0.3 mg) (FIG. 25,circles). The control group consisted of 5 mice and the treatment groupconsisted of 7 mice. Arthritis-associated joint swelling was determinedby caliper measurements taken every second day.

Study 2—Six-week-old male C57/B16 mice were injected with 150 μl of KBNsera on day 0 and day 2, and then were treated with either saline everysecond day (q2d) (FIG. 26, triangles), or one of the anti-Cad-11antibodies, H1M1 (FIG. 26, squares) or H14 (FIG. 26, circles), at 0.3mg/dose q2d. The control group consisted of 5 mice and the treatmentgroup consisted of 7 mice. Arthritis-associated joint swelling wasdetermined by caliper measurements taken every second day.

Results

Study 1—The H1M1 anti-Cad-11 antibody inhibited joint swelling relativeto the control mice. The greatest inhibition of arthritis-associatedjoint swelling was observed by dosing KBN-treated mice with 0.3 mg ofH1M1 antibody every second day (FIG. 25, circles).

Study 2—Both of the anti-Cad-11 antibodies inhibited joint swellingrelative to the control. In this study, the H14 antibody significantlydelayed the onset of arthritis compared to the control animals (FIG.27). All mice in the control group developed arthritis by day 3, whilethe H14-treated mice required 6 days before all of the animals developedarthritis.

These studies indicate that antibodies against the EC1 domain of humanCad-11 can inhibit the development and severity of arthritis in vivo.

Example 9 Generation of Antibodies Against Another EC1 Domain Peptide ofHuman Cadherin-11 Materials and Methods

Balb/c mice were immunized bi-weekly in the foot pad nine times over a 1month period with 0.01 mg of peptide V19-Y37 (VL VGRLH SDIDS GDGNI KY(SEQ ID NO:12)), corresponding to 19 amino acids of the human Cad-11 EC1domain, covalently linked to BSA. This peptide is referred to herein asPeptide 3. Spleens from the immunized mice were harvested and fused witha murine fusion partner P3×63-Ag8.653, to create antibody-producinghybridomas. These hybridomas were expanded and the anti-Cad-11antibody-containing media from the hybridomas were screened for theability to bind to a protein corresponding to the EC1-2 domain ofCad-11, which was produced in bacteria. The anti-Cad-11antibody-containing media from these Peptide 3 hybridomas were screenedconcurrently for the absence of binding to proteins encompassing theEC1-2 domains of Cad-8 and MN-Cadherin. 96-well EIA plates were coatedovernight at 4° C. with 0.05 ml of 0 to 300 mg/ml of one of each of theEC1-2 Cad proteins, or CHO cell produced EC1-Fc fusion protein, and thenwashed several times with saline buffer. Plates were then blocked using0.25 ml of casein-PBS buffer and subsequently washed several times withsaline buffer. Hybridoma media containing the Peptide 3 anti-Cad-11antibodies were incubated neat in each well for 1 hr at 22° C. and thenwashed twice with PBS-Tween (0.05%). 100 μl of a 1/1000 dilution of agoat anti-mouse IgG secondary antibody were added to each well,incubated for 30 min at 22° C., and then washed twice with PBS-Tween(0.05%). 100 μl/well of room temperature TMB(3,3′,5,5′-tetramethylbenzidine) reagent was added to each well andcolor was allowed to develop for 5 min at 22° C. The reaction wasstopped with 100 μl of room temperature 2N sulfuric acid and the platewas read at 450 nm on a Wallac 1420 microplate reader.

Media from the Peptide 3 hybridomas were also tested for the ability tobind to human Cad-11 protein expressed on cells. FrozenCad-11-expressing 431D cells were thawed and washed twice in HBSS withCa²⁺ and then resuspended at 10⁶ cells/ml in HBSS containing Ca²⁺. 10⁵cells/well were stained with either a 50% or 16% anti-Cad-11 antibodymedia for 45 min on ice, washed twice in HBSS containing Ca²⁺, and thenstained with a secondary goat anti-mouse IgG antibody conjugated withphytoerytherin at a concentration of 1% for 45 on ice and then washedagain twice in HBSS containing Ca²⁺. Cells were then resuspended in 400μl of HBSS containing Ca²⁺ and 1% formaldehyde and subsequently analyzedon a FACScalibur for PE positive cells.

Results

Anti-Cad-11 antibody-containing media from the Peptide 3 hybridomasbound to the Cad-11 EC1-2 protein and the EC1-Fc fusion protein (FIG.28, HL vs CAD11 and HL vs Cad11-EC1, respectively), but did not bindproteins containing the EC1-2 domains of Cad-8 and MN-Cad (FIG. 28, HLvs CAD8 and HL vs MNCAD, respectively). Control hybridoma media did notbind any of the cadherin proteins (FIG. 28, Media vs CAD11, Media vsCAD8, and Media vs MNCAD).

Anti-Cad-11 antibodies from the Peptide 3 hybridomas also bound to cellsexpressing human Cad-11 protein (FIG. 29, see arrow), but not tonon-Cad-11-expressing control cells that expressed Neos. This resultconfirmed the presence of anti-Cad-11 antibodies in the hybridomas thatrecognize both Peptide 3 and Cad-11-expressing cells in vitro.

The relevant teachings of all patents, published applications andreferences cited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A Cadherin-11 antagonist that specifically binds an EC1 domain of amammalian Cadherin-11 protein, wherein the Cadherin-11 antagonistinhibits aggregation of cells that express said mammalian Cadherin-11protein.
 2. (canceled)
 3. (canceled)
 4. An isolated antibody thatspecifically binds an EC1 domain of a mammalian Cadherin-11 protein,wherein the antibody inhibits aggregation of cells that express saidmammalian Cadherin-11 protein. 5-9. (canceled)
 10. A fusion proteincomprising at least a portion of a mammalian immunoglobulin protein anda portion of a human Cadherin-11 extracellular region that includesamino acids 54-90 of SEQ ID NO:2, wherein the portion of the humanCadherin-11 extracellular region does not include the entire humanCadherin-11 extracellular region consisting of amino acids 1-609 of SEQID NO:2. 11-18. (canceled)
 19. A method of treating an inflammatoryjoint disorder in a mammalian subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of aCadherin-11 antagonist that specifically binds an EC1 domain of amammalian Cadherin-11 protein, wherein the Cadherin-11 antagonistinhibits aggregation of cells that express said mammalian Cadherin-11protein in one or more joints of said subject.
 20. (canceled)
 21. Themethod of claim 19, wherein the inflammatory joint disorder is selectedfrom the group consisting of rheumatoid arthritis, osteoarthritis,psoriatic arthritis, Reiter's syndrome and ankylosing spondylitis. 22.The method of claim 19, wherein the inflammatory joint disorder isrheumatoid arthritis.
 23. The method of claim 19, wherein theCadherin-11 antagonist is an isolated antibody.
 24. (canceled)
 25. Themethod of claim 19, wherein the mammalian subject is a human. 26-28.(canceled)
 29. The method of claim 19, wherein the Cadherin-11antagonist inhibits migration, adhesion, invasion into cartilage, orintercellular signaling of cells that express said mammalian Cadherin-11protein in one or more joints of said subject.
 30. The method of claim19, wherein the Cadherin-11 antagonist inhibits induction of expressionor activity of an enzyme selected from the group consisting of acollagenase, a serine protease, and a matrix metalloproteinase in cellsthat express said mammalian Cadherin-11 protein in one or more joints ofsaid subject.
 31. The method of claim 19, wherein the Cadherin-11antagonist inhibits induction of expression or activity of a cytokine orgrowth factor selected from the group consisting of a IL-6, IL-8, RANKLand TRANCE in cells that express said mammalian Cadherin-11 protein inone or more joints of said subject.
 32. The method of claim 19, whereinthe Cadherin-11 antagonist is administered in combination with adisease-modifying anti-rheumatic drug. 33-36. (canceled)
 37. Apharmaceutical composition comprising the Cadherin-11 antagonist ofclaim 1, and a pharmaceutically-acceptable carrier.
 38. (canceled) 39.The pharmaceutical composition of claim 37, wherein the Cadherin-11antagonist is an isolated antibody. 40-52. (canceled)
 53. An isolatednucleic acid encoding the antibody of claim
 4. 54. (canceled)
 55. Anisolated cell expressing the antibody of claim
 4. 56. The cell of claim55, wherein said cell is a cell of hybridoma H1M1 (ATCC accession numberPTA-9699).
 57. The cell of claim 55, wherein said cell is a cell ofhybridoma H14 (ATCC accession number PTA-9701).
 58. The antibody ofclaim 4, wherein said antibody is produced by hybridoma H1M1 (ATCCaccession number PTA-9699).
 59. The antibody of claim 4, wherein saidantibody is produced by hybridoma H14 (ATCC accession number PTA-9701).60. (canceled)
 61. (canceled)